Method for producing hydrogen sulphide and the use thereof, in particular, for depolluting heavy metal-containing flows

ABSTRACT

The invention relates to using alcalophilic sulphate-reducing bacteria selected from at least one species of a Desulfohalobiaceae or  Desulfonatronum  species family or from species whose gene encoding for ribosomal PARN 16S has a homology equal to or greater than 97% with a corresponding gene of any species of the Desulfohalobiaceae or  Desulfonatronum  species family for producing a hydrogen sulphide substantially soluble in an aqueous medium.

The invention relates to a process for the production of hydrogensulphide and the use thereof, in particular for the decontamination ofeffluents containing heavy metals.

Heavy metals can be highly toxic to man and his environment. These heavymetals are not biodegradable, and hence they are of a cumulative nature.Thus more and more restrictive discharge standards have been imposed onindustrial activities discharging metals.

The processes most commonly used to separate the heavy metals containedin industrial effluents utilise the formation of metal hydroxides.However, these processes do not always satisfy the current environmentalstandards relating to the acceptable levels of dissolved metals ineffluents.

Another technology already in use involves the formation of metalsulphides. This requires the use of expensive synthetic polysulphides orthe formation of gaseous hydrogen sulphide by the action of hydrochloricacid on sodium sulphide.

Biological alternatives to this mode of treatment, again involving theproduction of gaseous hydrogen sulphide, have been proposed in the past.These biological alternatives are based on two main types of biologicalmechanisms capable of leading to the formation of hydrogen sulphide:

-   -   1) The assimilative reduction of sulphate is an anabolic        function which allows the majority of bacteria, fungi and plants        to incorporate sulphur into amino acids (cysteine, methionine        and cystine), vitamins (thiamine and biotin) and other        sulphurous molecules (ferredoxin for example . . . ) present in        the cells of these organisms. This reduction never leads        directly to the production of hydrogen sulphide. Nonetheless,        the latter is released indirectly during the fermentation of the        proteinaceous organic matter.    -   2) The dissimilative reduction of sulphates is carried out by        sulphate-reducing bacteria. In this anaerobic respiratory        process, the sulphate is used as a terminal electron acceptor        during the oxidation of hydrogen or of reduced organic compounds        such as acetate and propionate. During this metabolism, the        substrates are most often partially oxidised to acetate or, in        some cases, totally oxidised resulting in the formation of CO₂.

Many patents or patent applications relate to the use ofsulphate-reducing bacteria for the precipitation of metal ions as metalsulphides, in order to decontaminate effluents such as mine effluents orwaste waters.

Thus, the documents WO 80/02281 and U.S. Pat. No. 4,522,723, relate tothe use of bacteria of the Desulfovibrio or Desulfotomaculum type toreduce the levels of heavy metals in effluents. The document U.S. Pat.No. 4,108,722 envisages the injection of Vibrio and Desulfovibriobacteria into contaminated subterranean aquifer reservoirs. The documentU.S. Pat. No. 5,062,956 relates more specifically to hexavalent chromiumtreatment. Three other documents, U.S. Pat. No. 5,587,079, WO 97/29055and WO 02/06540 propose other processes for biological precipitation ofmetals, with the distinctive feature that the precipitation is carriedout sequentially, by varying the pH (mainly between 2.5 and 6.5) inorder for example first to precipitate copper, then zinc etc. . . . Thedocument WO 97/05237, which discloses the use of methylotrophicbacteria, i.e. which are capable of using methanol as the sole source ofcarbon, may also be cited. It must also be noted that systems ofco-culture of bacteria of different strains have also been envisaged,for example in the document U.S. Pat. No. 4,789,478 or the document EP 0692 458.

A disadvantage of the processes cited above resides in the fact thatsometimes considerable quantities of hydrogen sulphide in gaseous form(H₂S) are produced during these processes. This is particularly the casewhen it is desired to obtain maximal precipitation of the metalsinitially dissolved in the effluent to be decontaminated, which requiresthe use of the hydrogen sulphide in excess. Now hydrogen sulphide ingaseous form is toxic, corrosive, harmful to the environment andrequires an appropriate supplementary restrictive treatment.

One way of avoiding this disadvantage consists in producing dissolvedhydrogen sulphide (in the form of HS⁻) instead of gaseous hydrogensulphide. In order to do this, it is desirable that the pH of theculture solution in which the sulphide is produced by thesulphate-reducing bacteria be as high (basic) as possible. Now, in thegreat majority of the sulphate-reducing bacteria the use of a high pHadversely affects the sulphide production yield and can even be lethalto the culture.

One of the aspects of the invention is to produce essentially solublehydrogen sulphide, in good yield, by means of sulphate- orthiosulphate-reducing bacteria.

One of the other aspects of the invention is to propose new cultureconditions for certain sulphate-reducing bacteria, making it possible inparticular to make use of their thiosulphate-reducing properties.

One of the other aspects of the invention is to propose a process forthe decontamination of effluents containing heavy metals by means ofhydrogen sulphide produced by the culturing of sulphate- orthiosulphate-reducing bacteria.

Yet another aspect of the invention is to produce hydrogen sulphide forthe decontamination of effluents containing heavy metals, while avoidingthe undesirable presence of gaseous hydrogen sulphide.

These different aspects are obtained by using certain recentlydiscovered sulphate-reducing alkaliphilic bacteria, not until now usedto produce hydrogen sulphide or a fortiori for the decontamination ofeffluents laden with metals.

The invention thus relates to the use of alkaliphilic sulphate-reducingor thiosulphate-reducing bacteria selected from at least one species ofthe Desulfohalobiaceae family or of the Desulfonatronum genus or ofwhich the gene coding for the ribosomal RNA 16 S exhibits a homology ofat least 97% with the corresponding gene of any one of the species ofthe Desulfohalobiaceae family or of the Desulfonatronum genus, toproduce hydrogen sulphide in a form largely soluble in an aqueousmedium.

By “alkaliphilic bacteria” is meant bacteria whose life, growth, andvarious metabolic and enzymatic activities preferably take place at abasic pH.

A taxonomic, morphological and physiological description of the bacteriaused in the invention has been given in the following articles:

-   Pikuta et al., Desulfonatronum lacustre gen. nov. sp. nov.: a new    alkaliphilic sulphate-reducing bacterium utilizing ethanol,    Microbiology 67, 105;-   Zhilina et al., Desulfonatronovibrio hydrogenovorans gen. nov. sp.    nov., an alkaliphilic, sulphate-reducing bacterium; Int. J. Syst.    Bacteriol. Jan. 1997, p. 144;-   Pikuta et al., Desulfonatronum thiodismutans sp. nov., a novel    alkaliphilic, sulphate-reducing bacterium capable of    lithoautotrophic growth; Int. J. Syst. Evol. Microbiol. 53, 1327.

“Homology” is used to designate the proportion of identity between twonucleic acid sequences. This homology can be measured by seeking toalign the said sequences using an algorithm such as that defined inAltschul et al. (Nucl. Acid Res. 25:3389, 1997) or by using for examplethe software Clustal W, well known to the person skilled in the art anddescribed in Thompson et al. (Nucl. Acid Res. 22:4673, 1994).

By “form largely soluble” is meant a ratio of soluble hydrogen sulphideproduced to gaseous hydrogen sulphide produced greater than 1, and inparticular greater than 100.

As will be described in detail below, for the conditions for culturingthe sulphate-reducing bacteria according to the invention, novelsubstrates, namely formate and thiosulphate, are preferably used. Theformate serves as an energy source, while the thiosulphate serves as anelectron acceptor and source of sulphur: the strains of bacteria used inthe invention are thus thiosulphate-reducing as well as beingsulphate-reducing. This property of certain sulphate-reducing bacteriaof utilising thiosulphate as a substrate has not been used in thepreviously cited processes for metal decontamination.

The use of alkaliphilic sulphate- or thiosulphate-reducing bacteriaunder the conditions of the invention makes it possible to producehydrogen sulphide in a very largely soluble form, and in anexceptionally high yield.

Another decisive advantage of the invention is the possibility that itaffords of working with a pure culture pure without having to performany sterilisation, which makes considerable savings possible. In fact,the particular culture conditions which are used (high pH, mineralcontent, absence of O₂, high concentration of hydrogen sulphide . . . )ensure a strong selection pressure.

Advantageously, the use of alkaliphilic sulphate-reducing orthiosulphate-reducing bacteria according to the invention is carried outat a pH greater than or equal to about 9, in particular at a pH greaterthan or equal to about 9.5, in particular at a pH greater than or equalto about 10.

The proportion of the hydrogen sulphide produced according to theinvention which is in gaseous form (in other words the ratio of gaseoushydrogen sulphide produced to the dissolved hydrogen sulphide produced)in fact depends on the pH at which the hydrogen sulphide is produced. Atan acidic pH, the predominant form of the hydrogen sulphide is thegaseous form. At a pH of 7 there are about as many molecules of H₂S asHS⁻ ions. At a pH of 9, the proportion of gaseous hydrogen sulphide isonly about 1/100. At a pH of 9.5, the proportion of gaseous hydrogensulphide is only about 1/500. At a pH of 10, the proportion of gaseoushydrogen sulphide is only about 1/1000.

According to another advantageous implementation of the invention, theuse of alkaliphilic sulphate-reducing or thiosulphate-reducing bacteriatakes place in the form of culturing in the presence of an organiccompound serving as an electron donor, in particular formate, and in thepresence of a sulphurous compound serving as an electron acceptor, inparticular thiosulphate.

However, it is important to note that, depending on the bacterialstrains utilised, other substrates can serve for the production ofsulphide: for example sulphate, sulphite or sulphur as electronacceptor, and ethanol or dihydrogen as electron donor.

However that may be, the production of hydrogen sulphide is carried outin the invention via the reduction of a sulphurous compound. In the casewhere this sulphurous compound is thiosulphate (S₂O₃ ²⁻), the enzymaticreduction mechanisms involved are in particular based on thiosulphatesulphur transferase (or rhodanese) and on thiosulphate reductase.

In the first case, the outcome of the thiosulphate reduction reaction isequivalent to an oxidation of a thiol group by the thiosulphate, thesulphonyl part of which is reduced to sulphite:S₂O₃ ²⁻+2 RS⁻+H⁺→SO₃ ²⁻+R−SS−R+HS⁻

In the second case, the thiosulphate is cleaved to sulphite andsulphide, and the sulphite is then reduced to sulphide, with the finaloutcome:S₂O₃ ²⁻+4H₂→2HS⁻+3H₂O

Also a subject of the invention is a process for the production ofhydrogen sulphide in a form largely soluble in an aqueous mediumcomprising:

-   -   a stage of culturing alkaliphilic sulphate-reducing or        thiosulphate-reducing bacteria selected from at least one        species of the Desulfohalobiaceae family or of the        Desulfonatronum genus or of which the gene coding for the        ribosomal RNA 16 S exhibits a homology of at least 97% with the        corresponding gene of any one of the species of the        Desulfohalobiaceae family or of the Desulfonatronum genus, in        the presence of an organic compound serving as an electron        donor, in particular formate, and in the presence of a        sulphurous compound serving as an electron acceptor, in        particular thiosulphate, which leads to the formation of        hydrogen sulphide.

This bacterial culturing stage can for example be carried out in astandard reactor such as those available on the market for fermentation,on the pilot scale and on the industrial scale alike.

Advantageously, in the process for the production of hydrogen sulphideaccording to the invention, the culturing of the alkaliphilicsulphate-reducing or thiosulphate-reducing bacteria is carried out at apH greater than or equal to about 9, in particular at a pH greater thanor equal to about 9.5, in particular at a pH greater than or equal toabout 10.

If necessary, the pH can be measured and regulated by the addition ofacidic and/or basic substances.

According to a preferred implementation of the process for theproduction of hydrogen sulphide according to the invention, the bacteriaare selected such that they display a tolerance to hydrogen sulphide,the said tolerance being characterised in that the bacteria tolerateconcentrations of hydrogen sulphide at least greater than 20 mM.

By “tolerance” is meant the survival of the bacteria and the maintenanceof normal metabolic activity, which can be observed by the consumptionof the energy source.

This good tolerance of the bacteria in culture makes it possible toobtain solutions with a high concentration of hydrogen sulphide.

Advantageously, the bacteria used in the process for the production ofhydrogen sulphide according to the invention belong to the speciesDesulfonatronum lacustre.

Advantageously, the bacteria used in the process for the production ofhydrogen sulphide according to the invention belong to the speciesDesulfonatronovibrio hydrogenevorans.

According to an advantageous implementation of the invention, theculturing involved in the process for the production of hydrogensulphide is carried out on a support suitable for the growth of thebacteria, leading to the formation of a biofilm.

By “biofilm” is meant bacteria preferentially immobilised on a suitablesupport, of the pozzolane, Cloisonyl®, Bio-Net® or Sessil® type forexample, instead of a simple suspension of bacteria in solution.Culturing in biofilms makes it possible to obtain high concentrations ofbacterial cells locally and to produce hydrogen sulphide more rapidly.Moreover, few bacterial cells are removed from the reactor during thewithdrawal of the culture solution containing the hydrogen sulphide,since the bacteria are largely not in suspension.

According to a preferred implementation of the process for theproduction of hydrogen sulphide according to the invention, thiscomprises a sequence of the following two stages:

-   -   a first stage in which the culturing of the bacteria is carried        out under conditions appropriate for the growth of the said        bacteria and for the concomitant production of hydrogen        sulphide, and    -   a second stage in which the culturing of the bacteria is carried        out under conditions appropriate for the production of hydrogen        sulphide in the absence of growth of the said bacteria,

it being possible to repeat several times the sequence of the saidstages constituting one cycle if necessary.

By “growth of the said bacteria” is meant in particular theirmultiplication by cell division.

By “production of hydrogen sulphide in the absence of growth of the saidbacteria” is meant on the one hand the substantial arrest or markedslowing of growth, in other words of the multiplication of the bacteria,but also on the other hand the survival of these bacteria or at leastthe maintenance of enzymatic activity of bacterial origin causing theformation of hydrogen sulphide in the medium.

Preferably, the passage from one of the said stages to the other is inparticular carried out by means of addition of one or several acidic orbasic chemical substances, causing a change in the pH of the bacterialculture medium.

Particularly preferably, the said first stage is carried out at a pHranging from about 9 to about 10 and the said second stage is carriedout at a pH greater than about 10.

In fact, the bacteria used in the invention are capable of maintainingtheir hydrogen sulphide production activity at particularly extremealkaline pH, that is to say in particular at a pH greater than about 10.It is thus advantageous to culture the bacteria of the invention in aninitial period at their optimal culture pH, which in general liesbetween about 9 and about 10, so as to obtain the greatest possiblenumber of bacteria, then in a second period to pass to a pH greater thanabout 10 so as to continue to produce hydrogen sulphide, but underchemical conditions of the medium such that the ratio of the gaseoushydrogen sulphide produced to hydrogen sulphide produced is as low aspossible, and in particular less than 1/100.

According to a preferred implementation of the invention, the hydrogensulphide is produced at a concentration greater than or equal to about10 mM, in particular at a concentration greater than or equal to about20 mM, in particular at a concentration greater than or equal to about30 mM, in particular at a concentration greater than or equal to about40 mM.

The typical specific rate of production of hydrogen sulphide that can beattained according to the invention is at least 2.9 mmol HS⁻ g⁻¹ hr⁻¹and can range up to 5 mmol HS⁻ g⁻¹ hr⁻¹.

According to another preferred implementation of the invention, thehydrogen sulphide is produced by a culture of bacteria belonging to thespecies Desulfonatronum lacustre, the organic compound serving as anelectron donor being formate, and the sulphurous compound serving as anelectron acceptor being thiosulphate, and the bacteria are continuouslycultured on a biofilm at a pH greater than about 10.

Also a subject of the invention is a process for the decontamination ofan effluent containing one or more dissolved metals comprising:

-   -   a stage of production of hydrogen sulphide in a form largely        soluble in an aqueous medium by means of a culture of        alkaliphilic sulphate-reducing or thiosulphate-reducing bacteria        selected from at least one species of the Desulfohalobiaceae        family or of the Desulfonatronum genus or of which the gene        coding for the ribosomal RNA 16 S exhibits a homology of at        least 97% with the corresponding gene of any one of the species        of the Desulfohalobiaceae family or of the Desulfonatronum genus        in the presence of an organic compound serving as an electron        donor, in particular formate, and in the presence of a        sulphurous compound serving as an electron acceptor, in        particular thiosulphate, and    -   a stage of contacting the said effluent with the hydrogen        sulphide obtained in the preceding stage, resulting in the        reduction of the said dissolved metals and/or the precipitation        of the said dissolved metals in the form of metal sulphides, the        said contacting being carried out at a pH ranging from about 2        to about 12.

By “decontamination of an effluent containing one or more dissolvedmetals” is meant the significant reduction of the concentration of oneor more metals dissolved in the effluent, and in particular a reductionbelow the thresholds imposed by the various environmental standards fordischarges.

Examples of thresholds for metal concentrations in liquid dischargescurrently imposed by French legislation are as follows: 0.5 mg/L forcopper; 0.5 to 2 mg/L for zinc; 0.05 to 0.5 mg/L for arsenic; 0.05 to0.2 mg/L for cadmium; 0.1 mg/L for hexavalent chromium; 0.5 to 2 mg/Lfor tin; 0.03 to 0.05 mg/L for mercury; 0.5 to 2 mg/L for nickel and 0.1to 0.5 mg/L for lead. It must also be noted that the thresholds can varydepending on the industries involved, and that bylaws sometimes locallyset thresholds up to 100 times lower than the values cited above.

Now, with the exception of the special case of chromium, almost all themetals can be precipitated as metal sulphides by the action of hydrogensulphide and display lower solubility in the form of metal sulphidesthan in the form of metal hydroxides.

In fact the minimal solubilities observed, at various pH, for the metalhydroxides and sulphides are as follows:

-   -   5.2×10⁻² mg/L for arsenic sulphide (and no formation of arsenic        hydroxide);    -   6.7×10⁻¹⁰ mg/L for cadmium sulphide versus 2.3×10⁻⁵ mg/L for        cadmium hydroxide;    -   1.0×10⁻⁸ mg/L for copper sulphide versus 2.2×10⁻² mg/L for        copper hydroxide;    -   3.8×10⁻⁸ mg/L for tin sulphide versus 1.1×10⁻⁴ mg/L for tin        hydroxide;    -   2.1×10⁻³ mg/L for manganese sulphide versus 1.2 mg/L for        manganese hydroxide;    -   9.0×10⁻²⁰ mg/L for mercury sulphide versus 3.9×10⁻⁴ mg/L for        mercury hydroxide;    -   6.9×10⁻⁸ mg/L for nickel sulphide versus 6.9×10⁻³ mg/L for        nickel hydroxide;    -   3.8×10⁻⁹ mg/L for lead sulphide versus 2.1 mg/L for lead        hydroxide; and    -   2.3×10⁻⁷ mg/L for zinc sulphide versus 1.1 mg/L for zinc        hydroxide;

In the particular case of chromium in its hexavalent form, precipitationin the form of sulphide is not possible, but on the other hand thehydrogen sulphide makes it possible to reduce the Cr⁶⁺ ion to the Cr³⁺ion (Kim et al. 2001, Chromium VI reduction by hydrogen sulfide inaqueous media: stoichiometry and kinetics. Environ. Sci. Technol.35(11): 2219-2225), which is much less toxic and less soluble,particularly in its hydroxide form.

Further, the solubility of the metal hydroxides is generally minimal fora certain optimal pH value, whereas the solubility of the metalsulphides is typically a purely decreasing function of the pH, so thatit is always advantageous, solely from the point of view of thesolubility of the sulphides, to work at a pH as basic as possible.

In summary, the decontamination of effluents according to the inventionis thus particularly advantageous compared to processes making use of asimple precipitation in the form of hydroxides for:

-   -   arsenic, which cannot be insolubilised as hydroxide, and whose        insolubilisation as sulphide makes it possible to observe the        threshold values for discharge;    -   hexavalent chromium, which cannot precipitate efficiently as        hydroxide without having been previously reduced to trivalent        chromium by the hydrogen sulphide; and    -   manganese, zinc and lead, the precipitation of which as        sulphides makes it possible to observe the threshold values for        discharge, unlike the precipitation thereof as hydroxides.

Examples of effluents which can be treated according to the inventionare: effluents from the chemical, chemistry-related and petroleumindustries and in particular the coatings and pigment productionindustry; effluents from the mineral industry and in particular theglass industry (high discharge of lead); effluents from the engineeringand surface treatment sector; effluents from the iron and steel andmetallurgical sector (in particular for arsenic, chromium VI, lead andmanganese discharges); and effluents from the waste materials treatmentsector.

In the said process for the decontamination of an effluent containingone or more dissolved metals according to the invention, the hydrogensulphide production stage can be carried out in any of the mannersdescribed above.

According to a preferred implementation of the said process for thedecontamination of an effluent containing one or more dissolved metals,the stage of contacting the effluent with the hydrogen sulphide iscarried out at a neutral or basic pH.

This implementation makes it possible to minimise any release of gaseoushydrogen sulphide during the contacting stage and enables more completeprecipitation of the metal sulphides, which are less soluble at a highpH than at a low pH.

Advantageously, in the said process for the decontamination of aneffluent containing one or more dissolved metals, the production ofhydrogen sulphide and the contacting of the effluent with the hydrogensulphide are carried out in separate tanks, in particular respectively aculturing tank and a reaction tank, the said process comprising anintermediate stage between the hydrogen sulphide production stage andthe stage of contacting the effluent with the hydrogen sulphide, thesaid intermediate stage consisting in the injection of all or part ofthe hydrogen sulphide produced in the culturing tank into the reactiontank.

At the decontamination stage, in other words the contacting of thepolluted effluent and all or part of the hydrogen sulphide produced, inthe reaction tank, or after that said stage, it is possible to addcoagulating agents such as FeCl₃, in order to cause flocculation of theinsolubilised metal sulphides, then if necessary to pass the effluentinto a lamellar decanter in order to separate the precipitates aftersedimentation.

According to a particular implementation of the process for thedecontamination of an effluent containing one or more dissolved metalsaccording to the invention, a fraction of the effluent is injected intothe culturing tank.

This implementation can be described as production “with contacting” or“with partial contacting” of the effluent. In cases where thecomposition of the effluent is such that the effluent does notsignificantly reduce the capacity for the production of hydrogensulphide by the bacteria when these are cultured in contact with thateffluent, this implementation can present an economic advantage.

According to another particularly preferred, implementation of theprocess for the decontamination of an effluent containing one or moredissolved metals according to the invention, no fraction of the effluentis injected into the culturing tank.

This implementation can be described as production “in parallel with”the effluent. It is particularly advantageous, since the bacteriadestined to produce the hydrogen sulphide never come into contact withthe polluted effluent; now such contacting can impair the hydrogensulphide production capacity of the bacteria in a proportion which isdifficult to forecast, depending on their better or worse resistance tothe presence of dissolved metals in the culture medium.

Advantageously, the process for the decontamination of an effluentcontaining one or more dissolved metals according to the invention issuch that the stage of contacting the effluent with the hydrogensulphide in the reaction tank is carried out in the absence of a gaseousphase, so that there is no release of gaseous hydrogen sulphide.

By “in the absence of a gaseous phase” is meant: in the absence of anycontact with air or any other gas. According to the establishedterminology, this contacting therefore takes place in a “floodedmedium”. This characteristic is advantageous inasmuch as a release ofgaseous hydrogen sulphide is to be expected in the presence of a gaseousphase, and all the more since the pH in the reaction tank can be basicbut also neutral or acidic, in which case the chemical equilibriumbetween dissolved hydrogen sulphide and gaseous hydrogen sulphide isdisplaced in favour of the latter.

It should be noted that in the other previously cited implementationmodes of the decontamination process according to the invention, in caseof the presence of a gaseous. phase, the concentration of gaseoushydrogen sulphide possibly released during the contacting stage ispreferably less than 5 mg/m³.

As regards the culturing tank, this can contain a gaseous phase. In thatcase, the gaseous hydrogen sulphide possibly present in this gaseousphase can serve to reduce the oxidant compounds introduced into themedium. In case of gas overpressure, the gaseous hydrogen sulphide canbe neutralised by washing with caustic soda (NaOH). In any case, theconcentration of gaseous hydrogen sulphide released in the culturingtank is preferably less than 5 mg/m³.

According to a preferred implementation of the invention, the aforesaidprocess for the decontamination of an effluent containing one or moredissolved metals comprises the supplementary stages of:

-   -   measurement of the concentration of hydrogen sulphide produced        in the culturing tank,    -   estimation of the concentration of the metals or different        metals dissolved in the effluent to be decontaminated, and    -   adjustment of the quantity of solution containing hydrogen        sulphide having to be injected into the reaction tank on the        basis of the result of the said measurement and of the said        estimation.

In other words, according to this implementation it is possible to usethe process for the decontamination of an effluent according to theinvention in various applications, that is to say it is possible todecontaminate effluents having vary diverse characteristics in terms ofconcentrations of dissolved metals, by a simple and immediate adaptationof the process of the invention. In fact it suffices to have available asufficiently large production of hydrogen sulphide, then to adapt thequantity of hydrogen sulphide for contacting with the effluent to thenature of that effluent to be decontaminated.

Non-limiting examples of metal or metals dissolved in a pollutedeffluent that can be treated by means of the invention are: copper,zinc, arsenic, cadmium, chromium, particularly in its hexavalent form,tin, manganese, mercury, nickel, and lead.

Recovery, possibly selective, of the metal-containing precipitatesobtained by the process according to the invention, with a view to theirrecycling, is possible.

DESCRIPTION OF FIGURES

FIG. 1 a represents the growth of a culture of Desulfonatronum lacustreunder different culturing conditions. The x axis shows the time inhours. The y axis shows the optical density measurement at 580 nm.

FIG. 1 b represents the production of dissolved hydrogen sulphide duringculturing of Desulfonatronum lacustre under different culturingconditions. The x axis shows the time in hours. The y axis shows theconcentration of HS⁻ in mM.

For FIGS. 1 a and 1 b, the culturing condition components which vary asfollows: F=presence of formate; E=presence of ethanol; S=presence ofsulphate; T=presence of thiosulphate; YE=presence of yeast extract. Thedifferent sets of conditions are represented on both figures in thefollowing manner:

Symbols ×: E and T;

Symbols ◯: E and S;

Symbols +: E, T and YE;

Symbols □: E, S and YE;

Heavy continuous line: F and T;

Dashed line: F and S;

Dotted line: F, T and YE; and

Fine continuous line: F, S and YE.

FIG. 2 a represents the result of culturing Desulfonatronum lacustre ina “batch” type reactor at pH 9.5. The change in the concentration ofhydrogen sulphide is represented by a dotted line; that of theconcentration of formate is represented as a continuous line with ◯symbols and that of the optical density at 580 nm (indicative of theconcentration of bacteria) is represented as a continuous line with □symbols. The x axis corresponds to the time in hours. The left-hand yaxis corresponds to the concentrations in mM (for hydrogen sulphide andformate); and the right-hand y axis corresponds to the OD at 580 nm.

FIG. 2 b represents the result of culturing Desulfonatronum lacustre ina “batch” type reactor at pH 10. The change in the concentration ofhydrogen sulphide is represented by a dotted line; that of theconcentration of formate is represented as a continuous line with osymbols and that of the optical density at 580 nm (indicative of theconcentration of bacteria) is represented as a continuous line with □symbols. The x axis corresponds to the time in hours. The left-hand yaxis corresponds to the concentrations in mM (for hydrogen sulphide andformate); and the right-hand y axis corresponds to the OD at 580 nm. Itshould be noted that at t=92 hrs an injection of concentrated medium isperformed (see corresponding example below).

FIG. 3 represents the result of a heavy metals precipitation experimentaccording to the invention. Photo A represents a sample of 150 ml ofindustrial effluent essentially containing dissolved lead. Photo Brepresents the same sample immediately after injection of 5 ml ofculture medium obtained after 60 days of culturing of Desulfonatronumlacustre, containing more than 50 mM of hydrogen sulphide. Blackparticles of lead sulphide are visible.

EXPERIMENTAL SECTION

Bacterial Strains and Culture Media

Two bacterial strains are studied here: Desulfonatronum lacustre (pH:9.5) and Desulfonatrovibrio hydrogenevorans (pH: 9.5). Both strains arecultured at 37° C. The energy sources (electron donors) tested areethanol, and formate and the electron acceptors tested are sulphate andthiosulphate. The strains are described in detail in the examples below.The selection of the strains was performed using 5 ml cultures inHungate tubes.

The composition of the culture medium (called MLF) is as follows (ing/L):

NH₄Cl 1 K₂HPO₄ 0.3 KH₂PO₄ 0.3 CaCl₂•2H₂O 0.1 KCl 0.1 MgCl₂•6H₂O 0.1Cysteine 0.5 Na₂S 0.04% Widdel trace elements 1 mL Yeast extract 0.1

The sodium sulphide, the energy source, the electron acceptor and thebuffer defined below are added to the medium just before the inoculationof the latter. The sodium sulphide makes it possible greatly to diminishthe redox potential of the medium, thus creating conditions favourableto the growth of strictly anaerobic bacteria.

The buffer for the culturing performed at pH 9.5 is 1.6% Na₂CO₃.

Fermentation Device

The hydrogen sulphide production tests were performed by culturing ofthe selected strain in suspension in a “batch” type reactor (CHEMAP AG,Switzerland) of 20 L capacity.

The pH, temperature and stirring speed are regulated in a control boxadjacent to the fermenter (reactor). The pH is continuously regulated bymeans of a pH probe immersed in the culture medium. When a pH valuelower than the specified point is detected, a volume of caustic soda orcarbonate solution is injected into the fermenter by means of aperistaltic pump.

The temperature of the fermenter (37° C.) is regulated by circulation ofthermostatted water through the metal body of the apparatus.

The stirring (15 revolutions/minute) is carried out by a marine typepropeller.

The nitrogen feed is regulated by a ball flowmeter. A slightoverpressure is maintained in the fermenter by the incoming nitrogenflow and by immersion of the gas outlet in a 1N solution of causticsoda. This caustic soda solution also makes it possible to neutralisegaseous effluxes of H₂S that may be emitted during the fermentation.

A tap located at the bottom of the reactor tank makes it possible totake samples during the fermentation.

To start this, the reactor, containing 10 litres of MLF culture mediumand the thiosulphate, is initially sterilised with steam then cooledunder a current of nitrogen. The formate and an inoculum of 2 litres ofa culture at the end of the exponential growth phase are then injected.The pH is then adjusted by addition of sterile 40 mM carbonate anddeoxygenated.

Thereafter, the compounds added to the culture (caustic soda, carbonate,culture MLF medium) are not sterilised or deaerated beforehand.

Monitoring of Growth

The growth of the bacteria is monitored by means of a spectrophotometerby measurement of the optical density of the cultures at a wavelength of580 nm.

Previously performed studies made it possible to establish a linearrelationship between the cell concentration of the bacterium Thermotogaelfii (g of dry weight/litre), a thiosulphate-reducing bacterium, andthe optical density at 580 nm when the latter does not exceed 0.8:[Cells]=0.73×OD_(580 nm). This method is therefore applied here for theother bacteria.

The presence of precipitates in the cultures of Desulfonatronum lacustreimposes a mineralization of these necessary prior to the measurement ofthe optical density. This mineralization is carried out by addition of400 μL of 1M persulphuric acid to 4 mL of bacterial culture. Afterstirring, the mixture is allowed to stand for 2 minutes before readingof the optical density at 580 nm.

Colorimetric Assay of Sulphide

The dosage of dissolved sulphide is performed according to the method ofCord Ruwish (Cord Ruwish R., J. Microbiol. Methods. 4: 33-36, 1985):after sampling of 0.1 mL of culture medium, this is rapidly-mixed byvortexing with 4 mL of 5 mM CuSO₄, 50 mM HCl. The Bordeaux red-colouredcopper sulphide thus formed is titrated by measurement of the absorptionat 480 nm and comparison with a prerecorded standard curve.

The dosage of total sulphide is performed by prior basification of theculture medium to pH 12. This results in the dissolution of all of thesulphide which is then titrated as stated above.

Colorimetric Assay of Thiosulphate

The dosage of thiosulphate is performed according to the methoddescribed by Nor & Tabatabai (Nor Y. M§ and Tabatabai M. A., Anal. Lett.8: 537-547, 1975): 1 mL of sample is mixed with 1 ml of 0.1M KCN.S₂O₃ ²⁻→SO₃ ²⁻+CNS⁻

After 15 minutes, 2 mL of 0.33M CuCl₂ then 1 mL of Fe(NO₃)₃−HNO₃ areadded.CNS⁻+Fe³⁺→Fe−CNS

The mixture is adjusted to 25 mL with osmosed water, then, afterstirring and two minutes wait time, the quantity of sulphur present inthe sample is determined by measurement of the absorption of theiron-thiocyanate complex at 460 nm and comparison with a pre-recordedstandard curve.

Dosage of Sulphate

The sulphate is titrated according to the method of Tabatabai (TabatabaiM. A., Sulfur Institut Journal 10: 11-13, 1974): 1 mL of sample iswithdrawn under sterile conditions then acidified with 250 ml of 1N HClin an Eppendorf tube in order to remove sulphides. The cells are removedby centrifugation (14000 rpm, 3 minutes) and, 0.5 mL of supernatant istaken up in 4.5 mL of distilled water. Addition of 250 μL of BaCl₂ (1%w/v)−gelatine (0.3% w/v) makes it possible to precipitate the sulphatein the form of barium sulphate. After standing for 30 minutes, themixture is homogenised on the vortex and the absorption is measured at420 nm.

The standardisation is performed following the same protocol with 5 mLof distilled water and 250 μL of BaCl₂-gelatine solution.

The calibration is carried out on the basis of sulphate standards (1 mM,5 mM, 10 mM and 20 mM Na₂SO₄) to which the above protocol is applied.

Dosage of Sugars Organic Acids and Alcohols

The dosage of soluble metabolic products is performed by separation byhigh-pressure liquid phase chromatography (HPLC) on an ORH801 column(Interaction chemicals) with detection with an RID 6A differentialrefractometer (Shimadzu). The eluent is a filtered (0.65 μm Millipore)0.005N H₂SO₄ solution.

The samples are centrifuged (1300 revolutions·min⁻¹×15 min) before beinginjected.

Dosage of Gases

The dosage of gaseous products of the bacterial metabolism is performedby gas phase chromatography (GPC). The chromatograph (Chrompack CP 9000)is equipped with two columns mounted in series (Silicagel GC andMolecular Sieve 5A). Detection is carried out by thermal conductivityusing a catharometer. The carrier gas is helium at 2 bars, and thedetector filament current is 70 mA. The gas injections (0.1 mL) areperformed using a syringe fitted with a valve of the Minimert™ typemaking it possible to maintain the pressure of the sample.

EXAMPLES Description of Desulfonatronum lacustre

This is a sulphate- and thiosulphate-reducing, alkaliphilic,salt-tolerant and chemolithotrophic bacterium.

Morphology: vibrio, 0.7-0.9×2-3 μm, isolated, in pairs or in spiralchains, mobile by means of a polar flagellum. Gram negative.Multiplication by binary fission. Colony in agar: lenticular, 0.5-2 mmØ, yellowish then brown, translucent, with whole edge.

Metabolism: non-fermentative

Electron acceptors: sulphate, sulphite, thiosulphate.

Dismutation of thiosulphate to sulphide+sulphate.

Electron donors: H₂−CO₂, formate, ethanol→acetate.

No growth on acetate, propionate, butyrate, pyruvate, lactate, malate,fumarate, succinate, methanol, glycerol, choline, betaine, casaminoacids, yeast extract, glucose, fructose, mannose, xylose or rhamnose.

Syntrophy with Spirochaeta alcalica or Desulfonatronovibriohydrogenovorans.

Presence of cyt c, absence of desulphoviridine.

Growth stimulated by yeast extract or acetate.

Physiology:

-   -   T optimum=37-40° C. (20-45° C.).    -   pH optimum=9.5 (8-10).    -   NaCl optimum=0% w/v (0-10% w/v).

Dependent on sodium ions and carbonate.

DNA: 57.3 mol % G+C

RNA 16S sequence: Y14594

Typical strain Z-7951 (DSM 10312).

Origin: sediment from Lake Khadyn.

Reference: Pikuta E V, Zhilina T N, Zavarzin G A, Kostrikina N A, OsipovG A, Rainey F A (1998) Desulfonatronum lacustre gen. nov., sp. nov., anew alkaliphilic sulphate-reducing bacterium utilizing ethanol.Microbiology (Engl. Tr. Mikrobiologiya) 67, 123-131.

Description of Desulfonatrovibrio hydrogenevorans

Sulphate- and thiosulphate-reducing, alkaliphilic, weakly halophilicbacterium.

Morphology: vibrio, 0.5×1.5-2 μm, 1 polar flagellum, filamentousappendages, isolated or in pairs or in short chains, Gram negative.

Metabolism: lithoheterotrophic

Electron acceptors: sulphate, sulphite, thiosulphate.

Electron donors: H₂+CO₂, formate.

Carbon sources: acetate with yeast extract or vitamins.

Formation of sulphide on dimethyl sulphoxide with no growth.

Growth inhibited by sulphur.

Disproportionation of thiosulphate to sulphate and sulphide.

No desulphoviridine.

Physiology: alkaliphilic weakly halophilic

T optimum: 37° C. (15-43° C.)

pH optimum: 9.5-9.7 (8-10.2)

NaCl optimum: 3% (1-12%)

t_(1/2) optimum=26.5 hrs on sulphate, 20.1 hr on thiosulphate.

Dependent on Na⁺

DNA: 48.6 mol % G+C (Tm)

RNA 16S sequence: X99234

Typical strain: Z-7935T (DSM 9292 T).

Origin: sediments of alkaline lakes (equatorial Lake Magadi)

Reference: Zhilina T N, Zavarzin G A, Rainey F A, Pikuta E N, Osipov GA, Kostrikina N A (1997) Desulfonatronovibrio hydrogenovorans gen. nov.,sp. nov., an alkaliphilic, sulphate-reducing bacterium. Int. J. Syst.Bacteriol. 47, 144-149.

Determination of the Optimal Culture Conditions for the Production ofHydrogen Sulphide by Desulfonatronum lacustre

Reference is made here to FIG. 1 a, which represents the measurement ofthe optical density with the passage of time, which is directly relatedto the concentration of the bacteria, as a function of time; and to FIG.1 b, which represents the measurement of the concentration of dissolvedhydrogen sulphide as a function of time.

Different culture conditions are tested:

F: presence of formate; E: presence of ethanol; S: presence of sulphate;T: presence of thiosulphate; YE: presence of yeast extract (Panreac,Spain).

The experiments were performed in duplicate. FIGS. 1 a and 1 b representthe mean of the results derived from the measurements of the OD at 580nm and of the hydrogen sulphide dosage for the different cultures.

Examination of these results shows that:

-   -   the use of formate makes it possible to obtain higher        concentrations of hydrogen sulphide than the use of ethanol;    -   cultures in the presence of thiosulphate make it possible to        obtain higher concentrations of hydrogen sulphide than in the        presence of sulphate;    -   the addition of yeast extract to the culture medium does not        make it possible to obtain results significantly different from        those obtained without that addition; the reason for this is        that yeast extract is present in trace amounts in the inoculum        of bacteria;    -   Desulfonatronum lacustre reduces thiosulphate by oxidation of        formate producing high concentrations (about 22 mM) of hydrogen        sulphide;    -   the rate of production of hydrogen sulphide is 0.122 mM/hr.

Desulfonatronum lacustre is thus suitable for the production of hydrogensulphide and was retained for the continuation of the experiments owingto:

-   -   its ability to produce high concentrations of hydrogen sulphide;    -   its rate of production of hydrogen sulphide;    -   its alkaliphilia enabling greater solubility of the hydrogen        sulphide and a selection pressure for maintenance of the strain        in culture. The high pH values eliminate exogenous contamination        problems.

Production of Hydrogen Sulphide by Desulfonatronum lacustre in “Batch”Reactor

1. Importance of Carbonate for Growth

The implementation of the first cultures of Desulfonatronum lacustre ina batch reactor made it possible to confirm the dependence of thisstrain on carbonate. In fact, in the absence of sodium carbonate in theculture medium, no growth of this bacterium was observed for a week. Theaddition of a concentration of sodium bicarbonate less than 16.6 g/ldoes not enable growth either. It has been postulated that carbonate isinvolved in the dissociation equilibrium of formate via the CO₂concentration of the gaseous phase.

2. Growth and Production at pH 9.5

A culture of Desulfonatronum lacustre was carried out in the firstinstance by inoculation of 10 liters of medium with an inoculumconsisting of two liters of a culture at the end of the exponentialgrowth phase. The energy source is represented by formate (74 mM) andthe terminal electron acceptor is thiosulphate (20 mM). The adjustmentof the pH to 9.5 is initially performed with sodium carbonate. The pH isthen regulated by injection of 4N caustic soda solution. The results ofthe experiment are shown on FIG. 2 a.

The growth of Desulfonatronum lacustre follows a typical curve ofsigmoidal shape: after a latency phase (t<50 hrs), the bacteria enter aphase of exponential growth (50 hrs≦t≦200 hrs), then retardation phaseappears (t>200 hrs). A stationary phase is observed from t=200 hrs. Theformate being monitored in real time, the energy source was replenishedfor a second series of experiments at pH 9.5 after its concentrationreached 0 mM.

The optical density of the culture is remarkably low and only increasesvery slightly on addition of formate: a growth limitation seems to be inoperation. The generation time of Desulfonatronum lacustre under theexperimental conditions is 71.45 hrs at pH 9.5. This value isremarkable, even for a thiosulphate-reducing bacterium. By comparison,the generation time of Desulfonatronum lacustre under the sameexperimental conditions (medium, formate, thiosulphate, yeast extract,temperature, pH) but during its culturing in a Hungate tube (5 mL ofmedium) is 84.52 hrs. It thus appears that culturing in a fermenterwhere stirring and continuous regulation of the pH are maintained andwhere the liquid volume/gas volume ratio is higher makes it possible todecrease the generation time of this bacterium.

Observation of the consumption of formate shows that this is correlatedwith the bacterial growth. Low during the initial latency phase, theconsumption of formate increases during the exponential growth phasethen slows during the stationary phase. However, it is noted that theconsumption of formate is maintained, which is consistent with theobservation of the maintenance of hydrogen sulphide production activityby the bacteria in the stationary state.

By correlation between the quantity of formate consumed and that ofhydrogen sulphide produced, it is deduced that under the experimentalconditions the formation of one mole of hydrogen sulphide requires theoxidation of 2.83 moles of formate at pH 9.5.

After establishment of a biomass inside the reactor and exhaustion ofthe energy source, a correlation was established between the opticaldensity of the culture at 580 nm and the biomass. Thus for anOD_(580 nm) of 0.465, a dry weight of 67.6 mg/L was determined. Assumingthat the relationship biomass=ft(OD_(580 nm)) is linear, we have:Biomass (mg/L)=145.37×OD _(580 nm)

It was possible to determine the specific rate of production of hydrogensulphide. The specific rate of production of hydrogen sulphide at pH 9.5under the experimental conditions is 3.318 mmol HS⁻ g⁻¹ hr⁻¹.

3. Growth and Production at pH 10

Growth of Desulfonatronum lacustre was then carried out at pH 10 byaddition of concentrated culture medium to the reactor and alteration ofthe specified value for the pH. The results of this experiment arepresented in FIG. 2 b. At t=92 hrs, the culture medium was enriched byaddition of 1 L of concentrated culture medium (equivalent to 8 litresof standard medium) in order to replenish the energy source theconcentration of which was decreasing markedly. This addition caused adecrease in the concentration of hydrogen sulphide and a decrease in theOD_(580 nm).

It can be observed that the growth of Desulfonatronum lacustre ismaintained at pH 10. This is still correlated with the oxidation of theenergy source, the formate. It is however more than twice as slow as atpH 9.5. In fact the generation time of Desulfonatronum lacustre underour experimental conditions is 141.45 hrs at pH 10.

It can be noted that the concentration of hydrogen sulphide can attainthe value of 32 mM. This high concentration of sulphide can be the causeof the decrease in the rate of growth of Desulfonatronum lacustre. Thisstrain can however be considered exceptionally tolerant to sulphides inview of the concentrations of hydrogen sulphide encountered in theculture medium.

By correlation between the quantity of formate consumed and that ofhydrogen sulphide produced, we deduce that increasing the pH of theculture medium to a value of 10 does not change the energy yield of theformation of hydrogen sulphide. In fact, the values at pH 9.5 and pH 10are almost identical: the formation of one mole of hydrogen sulphiderequires the oxidation of 2.66 moles of formate at pH 10.

However, the specific rate of production of hydrogen sulphide decreasesslightly (11%) with the change in the pH from 9.5 to 10. The specificrate of production of hydrogen sulphide at pH 10 under our experimentalconditions is 2.974±0.635 mmol HS⁻ g⁻¹ hr⁻¹.

This decrease in the activity of Desulfonatronum lacustre with thechange from pH 9.5 to pH 10 is not in agreement with the results ofPikuta et al. who observed a 50% decrease in the sulphidogenic activityof this strain with the change from pH 9.5 to pH 10.

During culturing performed at pH 10, the medium added to the reactor wasneither sterilised nor deaerated. Microscopic examination made itpossible to check that only Desulfonatronum lacustre was able tomaintain itself under its growth-restrictive conditions (high pH,inorganic medium, high concentration of sulphides). Further, the highconcentration of sulphides in the reactor makes it possible to maintaincomplete absence of oxygen therein, as is shown by the maintenance of ahigh concentration of hydrogen sulphide.

Optimisation of the Culture Medium

Cultures were performed in Hungate tubes in different culture mediaderived from the standard culture medium MLF. Depending on the cultureseries, the cysteine, the sodium sulphide, the acetate, the yeastextract or the trace elements were omitted. The first inoculation ofDesulfonatronum lacustre was performed from a pre-culture on rich MLFmedium. Thereafter, for the second (t=10 days) and third (t=20 days)subcultures, the inoculum was provided by the culture previouslyperformed on the same type of medium. These subcultures in series makeit possible to ensure the absence of the desired compound through theeffect of the dilutions.

It should be recalled that in the medium MLF, the cysteine and thesodium sulphide are added as reducing agents making it possible tofavour the establishment of a low redox potential favourable to thegrowth of Desulfonatronum lacustre. The acetate is added to provide acomplementary source of carbon readily available to the bacterialstrain, which should allow it to initiate its growth. The yeast extractis a source of amino acids and of inorganic growth factors for thisheterotrophic strain and the solution of Widdel trace elements providesmany metals that may be involved in the bacterial metabolism.

The results derived from the third subculturing of the differentalternative culture media are shown in Table 1 below. From thecomparison of these results, it can be noted that the three lowestgrowths and the three lowest hydrogen sulphide production levels arecorrelated with the absence of yeast extract (media B, D and I).

A low concentration of yeast extract (0.1 g/L) appears indispensable forgood growth of Desulfonatronum lacustre and high production of hydrogensulphide by this strain. To a lesser extent, the omission of thesolution of Widdel trace elements appears to limit the production ofhydrogen sulphide by Desulfonatronum lacustre. The role of these traceelements can be explained by the presence of metals in the haems of theenzymes involved in the sulphate respiration in the sulphate-reducingbacteria. These elements could be supplied by the impurities encounteredin products of industrial quality and which are not found in theproducts of “analytical” quality used in the laboratory.

TABLE 1 Concentrations Compounds (mg/l) A B C D E F G H I Cysteine 0.5 XX X X X X Sodium sulphide 0.4 X X X X X X Sodium acetate  0.16 X X X X(2 mM) Yeast extract 0.1 X X X X X X Widdel 1 ml X X X X X X traceelements Results of 3^(rd) OD_(580 nm) 0.23 0.18 0.25 0.18 0.3 0.22 0.230.24 0.2 subculturing * HS⁻ 9.35 6.45 10.5 2.35 14 10.9 8.85 12.5 1.9 *the results are the mean of two series of experiments.

Precipitation of Copper by Hydrogen Sulphide

The dosage of the dissolved hydrogen sulphide is performed bymeasurement of the absorbance of the Bordeaux red-coloured coppersulphide formed by the action of the hydrogen sulphide on coppersulphate. The reaction involved in this dosage was exploited toillustrate the capabilities of hydrogen sulphide for the separation ofdissolved metals in an effluent.

The copper sulphide is formed instantaneously on mixing of the hydrogensulphide and the dissolved copper with stirring. The copper sulphidethus formed precipitates rapidly, in a manner visible to the naked eye.When the mixture is allowed to stand for 5 minutes, the major part ofthe precipitate is found at the bottom of the test-tube.

The addition of a coagulant such as FeCl₃ makes it possible toprecipitate the whole of the copper. The precipitation of metals in theform of sulphides by the use of the hydrogen sulphide present incultures of sulphate- and thiosulphate-reducing bacteria has beenconfirmed.

Decontamination of an Industrial Effluent

A precipitation of heavy metals contained in an actual industrialeffluent provided by the company X, situated in the Vaucluse andconfronted with contamination of its effluents with the lead which itconverts in the context of its activity of accumulator production, isperformed.

Desulfonatronum lacustre is cultured at 37° C. for more than 2 months in2 litres of MLF medium. The initial pH of the culture is 9.5. The energysource utilised is formate, and the sulphur source is thiosulphate.

The polluted effluent is drawn off upstream of the currently existingtreatment works.

A measurement of the concentration of hydrogen sulphide is firstpreformed. After dilution of the culture medium to ¼, the measurementgives a concentration of 4×12.9=51.6 mM. This exceptional concentrationof hydrogen sulphide in a culture medium is due to a particularly longculturing time (greater than 60 days).

Analysis of the final pH of the medium shows that this is 8.8. The fallin the pH from 9.5 to 8.8 in spite of the presence of a carbonate bufferconfirms the generation of a large quantity of acid during the culturingof Desulfonatronum lacustre.

This concentration of hydrogen sulphide greater than 50 mM in theculture medium shows that Desulfonatronum lacustre is capable oftolerating particularly high concentrations of sulphides.

5 ml of culture medium containing 51.6 mM of hydrogen sulphide arewithdrawn by means of a syringe and injected into 150 ml of contaminatedeffluent. The effluent before injection is shown on photo A in FIG. 3,and the effluent immediately after injection is shown on photo B in FIG.3.

Following the injection of the hydrogen sulphide into the effluent,particles of lead sulphides, identified by the characteristic lustrousblack colour, appear and rapidly sediment.

The injection of culture medium containing hydrogen sulphide produced bythe metabolism of Desulfonatronum lacustre thus makes it possibleinstantaneously to effect the precipitation of the lead and itsseparation from the industrial effluent.

1. Process for the production of hydrogen sulphide in a form largelysoluble in an aqueous medium, the process comprising: a stage ofculturing alkaliphilic sulphate-reducing or thio-sulphate reducingbacteria selected from at least one species of the Desulfohalobiaceaefamily or of the Desulfonatronum genus, or of which the gene coding forthe ribosomal RNA 16 S exhibits a homology of at least 97% with thecorresponding gene of any one of the species of the Desulfohalobiaceaefamily or of the Desulfonatronum genus, in the presence of an organiccompound serving as an electron donor, and in the presence of asulphurous compound serving as an electron acceptor, which leads to theformation of hydrogen sulphide, and a sequence of the following twostages: a first stage in which the culturing of the bacteria is carriedout under conditions appropriate for the growth of the bacteria and forthe concomitant production of hydrogen sulphide, and a second stage inwhich the culturing of the bacteria is carried out under conditionsappropriate for the production of hydrogen sulphide in the absence ofgrowth of the said bacteria, and optionally repeating the sequence ofthe stages constituting one cycle several times if necessary.
 2. Processfor the production of hydrogen sulphide according to claim 1, whereinthe organic compound serving as an electron donor is formate and thesulphurous compound serving as an electron acceptor is thiosulphate. 3.Process for the production of hydrogen sulphide according to claim 1,wherein the culturing of the alkaliphilic sulphate-reducing orthiosulphate-reducing bacteria is carried out at a pH greater than orequal to
 9. 4. Process for the production of hydrogen sulphide accordingto claim 1, wherein the culturing of the alkaliphilic sulphate-reducingor thiosulphate-reducing bacteria is carried out at a pH greater than orequal to 9.5.
 5. Process for the production of hydrogen sulphideaccording to claim 1, wherein the culturing of the alkaliphilicsulphate-reducing or thiosulphate-reducing bacteria is carried out at apH greater than or equal to
 10. 6. Process for the production ofhydrogen sulphide according to claim 1, wherein the bacteria have atolerance to hydrogen sulphide, and the bacteria tolerate concentrationsof hydrogen sulphide at least greater than 20 mM.
 7. Process for theproduction of hydrogen sulphide according to claim 1, wherein thebacteria belong to the species Desulfonatronum lacustre.
 8. Process forthe production of hydrogen sulphide according to claim 1, wherein thebacteria belong to the species Desulfonatronovibrio hydrogenevorans. 9.Process for the production of hydrogen sulphide according to claim 1,wherein the culturing is carried out on a support suitable for thegrowth of the bacteria, leading to the formation of a biofilm. 10.Process for the production of hydrogen sulphide according to claim 1,wherein the passage from one stage to the other is carried out byaddition of one or several acidic or basic chemical substances resultingin a change in the pH of the culture medium of the bacteria.
 11. Processfor the production of hydrogen sulphide according to claim 1, whereinthe first stage is carried out at a pH ranging from 9 to 10 and thesecond stage is carried out at a pH greater than
 10. 12. Process for theproduction of hydrogen sulphide according to claim 1, wherein thehydrogen sulphide is produced at a concentration greater than or equalto 10 mM.
 13. Process for the production of hydrogen sulphide accordingto claim 1, wherein the hydrogen sulphide is produced at a concentrationgreater than or equal to 20 mM.
 14. Process for the production ofhydrogen sulphide according to claim 1, wherein the hydrogen sulphide isproduced at a concentration greater than or equal to 30 mM.
 15. Processfor the production of hydrogen sulphide according to claim 1, whereinthe said bacteria belong to the species Desulfonatronum lacustre, theorganic compound serving as an electron donor is formate, the sulphurouscompound serving as an electron acceptor is thiosulphate and thebacteria are continuously cultured on a biofilm and the pH is greaterthan 10.